The invention relates to a simple, and preferably cost effective, method for immobilizing nucleic acid molecules onto a solid support. The invention further concerns the use of such immobilized molecules in nucleic acid hybridization assays, sequencing by hybridization assays, and genetic analyses and combinatorial analyses involving nucleic acids or proteins for screening applications.
The analysis of the structure, organization and sequence of nucleic acid molecules is of profound importance in the prediction, diagnosis and treatment of human and animal disease, in forensics, in epidemiology and public health, and in the elucidation of the factors that control gene expression and development. Methods for immobilizing nucleic acids are often important in these types of analyses. Three areas of particular importance involve hybridization assays, nucleic acid sequencing, and the analysis of genomic polymorphisms.
The capacity of a nucleic acid xe2x80x9cprobexe2x80x9d molecule to hybridize (i.e. base pair) to a complementary nucleic acid xe2x80x9ctargetxe2x80x9d molecule forms the cornerstone for a wide array of diagnostic and therapeutic procedures.
Hybridization assays are extensively used in molecular biology and medicine. Methods of performing such hybridization reactions are disclosed by, for example, Sambrook, J. et al. (In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), Haymes, B. D., et al. (In: Nucleic Acid Hybridization, A Practical Approach , IRL Press, Washington, D.C. (1985)) and Keller, G. H. and Manak, M. M. (In: DNA Probes, Second Edition, Stockton Press, New York, N.Y. (1993)) which references are incorporated herein by reference.
Many hybridization assays require the immobilization of one component. Nagata et al. described a method for quantifying DNA which involved binding unknown amounts of cloned DNA to microtiter wells in the presence of 0.1 M MgCl2 (Nagata et al., FEBS Letters 183: 379-382, 1985). A complementary biotinylated probe was then hybridized to the DNA in each well and the bound probe measured colorimetrically. Dahlen, P. et al. have discussed sandwich hybridization in microtiter wells using cloned capture DNA adsorbed to the wells (Dahlen, P. et al., Mol. Cell. Probes 1: 159-168, 1987). An assay for the detection of HIV-1 DNA using PCR amplification and capture hybridization in microtiter wells has also been discussed (Keller, G. H. et al., J. Clin. Microbiol. 29: 638-641, 1991). The NaCl-mediated binding of oligomers to polystyrene wells has been discussed by Cros et al. (French patent no. 2,663,040) and very recently by Nikiforov et al. (PCR Methods Applic. 3: 285-291, 1994). The cationic detergent-mediated binding of oligomers to polystyrene wells has very recently been described by Nikiforov et al., Nucleic Acids Res. 22: 4167-4175.
Many genetic diseases and traits (i.e. hemophilia, sickle-cell anemia, cystic fibrosis, etc.) reflect the consequences of mutations that have arisen in the genomes of some members of a species through mutation or evolution (Gusella, J. F., Ann. Rev. Biochem. 55:831-854 (1986)). In some cases, such polymorphisms are linked to a genetic locus responsible for the disease or trait; in other cases, the polymorphisms are the determinative characteristic of the condition.
Such single nucleotide polymorphisms differ significantly from the variable nucleotide type polymorphisms (xe2x80x9cVNTRsxe2x80x9d), that arise from spontaneous tandem duplications of di- or tri-nucleotide repeated motifs of nucleotides (Weber, J. L., U.S. Pat. No. 5,075,217; Armour, A. J. et al., FEBS Lett. 307:113-115 (1992); Jones, L. et al., Eur. J. Haematol. 39:144-147 (1987); Horn, G. T. et al. PCT Application WO91/14003; Jeffreys, A. J., U.S. Pat. No. 5,175,082); Jeffreys. A. J. et al., Amer. J. Hum. Genet. 39:11-24 (1986); Jeffreys. A. J. et al., Nature 316:76-79 (1985); Gray, I. C. et al., Proc. R. Acad. Soc. Lond. 243:241-253 (1991); Moore, S. S. et al., Genomics 10:654-660 (1991); Jeffreys, A. J. et al., Anim. Genet. 18:1-15 (1987); Hillel, J. et al., Anim. Genet. 20:145-155 (1989); Hillel, J. et al., Genet. 124:783-789 (1990)), and from the restriction fragment length polymorphisms (xe2x80x9cRFLPsxe2x80x9d) that comprise variations which alter the lengths of the fragments that are generated by restriction endonuclease cleavage (Glassberg, J., UK patent application 2135774; Skolnick, M. H. et al., Cytogen. Cell Genet. 32:58-67 (1982); Botstein, D. et al., Ann. J. Hum. Genet. 32:314-331 (1980); Fischer, S. G. et al. (PCT Application WO90/13668); Uhlen, M., PCT Application WO90/11369)).
Because single nucleotide polymorphisms constitute sites of variation flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single nucleotide present at the site of variation; it is unnecessary to determine a complete gene sequence for each patient. Several methods have been developed to facilitate the analysis of such single nucleotide polymorphisms.
Mundy, C. R. (U.S. Pat. No. 4,656,127), for example, discusses a method for determining the identity of the nucleotide present at a particular polymorphic site that employs a specialized exonuclease-resistant nucleotide derivative. A primer complementary to the allelic sequence immediately 3xe2x80x2 to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. The Mundy method has the advantage that it does not require the determination of large amounts of extraneous sequence data. It has the disadvantages of destroying the amplified target sequences, and unmodified primer and of being extremely sensitive to the rate of polymerase incorporation of the specific exonuclease-resistant nucleotide being used.
Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087) discuss a solution-based method for determining the identity of the nucleotide of a polymorphic site. As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3xe2x80x2 to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.
An alternative method, known as Genetic Bit Analysis(trademark) or GBA(trademark) is described by Goelet, P. et al. (PCT Appln. No. 92/15712). The method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3xe2x80x2 to a polymorphic site. The labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087) the method of Goelet, P. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase. It is thus easier to perform, and more accurate than the method discussed by Cohen.
Cheesman, P. (U.S. Pat. No. 5,302,509) describes a method for sequencing a single stranded DNA molecule using fluorescently labeled 3xe2x80x2-blocked nucleotide triphosphates. An apparatus for the separation, concentration and detection of a DNA molecule in a liquid sample has been recently described by Ritterband, et al. (PCT Patent Application No. WO95/17676).
An alternative approach, the xe2x80x9cOligonucleotide Ligation Assayxe2x80x9d (xe2x80x9cOLAxe2x80x9d) (Landegren, U. et al., Science 241:1077-1080 (1988)) has also been described as capable of detecting single nucleotide polymorphisms. The OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA. In addition to requiring multiple, and separate, processing steps, one problem associated with such combinations is that they inherit all of the problems associated with PCR and OLA.
Recently, several primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Komber, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvxc3xa4nen, A.-C., et al., Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyrxc3xa9n, P. et al., Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA(trademark) in that they all rely on the incorporation of labeled deoxynucleotides to discriminate between bases at a polymorphic site. In such a format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvxc3xa4nen, A.-C., et al., Amer. J. Hum. Genet. 52:46-59 (1993)). Such a range of locus-specific signals could be more complex to interpret, especially for heterozygotes, compared to the simple, ternary (2:0, 1:1, or 0:2) class of signals produced by the GBA(trademark) method. In addition, for some loci, incorporation of an incorrect deoxynucleotide can occur even in the presence of the correct dideoxynucleotide (Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989)). Such deoxynucleotide misincorporation events may be due to the Km of the DNA polymerase for the mispaired deoxy-substrate being comparable, in some sequence contexts, to the relatively poor Km of even a correctly base paired dideoxy-substrate (Kornberg, A., et al., In: DNA Replication, Second Edition (1992), W.H. Freeman and Company, New York; Tabor, S. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:4076-4080 (1989)). This effect would contribute to the background noise in the polymorphic site interrogation.
Several of the above-described methods involve procedures in which one or more of the nucleic acid reactants are immobilized to a solid support. Currently, 96-well polystyrene plates are widely used in solid-phase immunoassays, and several PCR product detection methods that use plates as a solid support have been described. The most specific of these methods require the immobilization of a suitable oligonucleotide probe into the microtiter wells followed by the capture of the PCR product by hybridization and colorimetric detection of a suitable hapten. It would be desirable to have an improved immobilization method that could be used to bind oligonucleotides to polystyrene such that their capacity to be used for hybridization, sequencing, or polymorphic analysis would be retained, and which would be rapid, convenient to use and inexpensive. The present invention provides such an improved method.
The means by which macromolecules bind non-covalently to polystyrene and glass surfaces is not well understood. Nevertheless, these adsorption phenomena have proven to be important in the development and manufacturing of immunoassays and other types of diagnostic tests where one component needs to be immobilized.
Polystyrene is a very hydrophobic material because it normally contains no hydrophilic groups. Microtiter plate manufacturers have developed methods of introducing such groups (hydroxyl, carboxylate and others) onto the surface of microwells to increase the hydrophilic nature of the surface. Theoretically, this allows macromolecules to bind through a combination of hydrophobic and hydrophilic interactions (Baier et al., Science 162: 1360-1368 (1968); Baier et al., J. Biomed. Mater. Res. 18: 335-355 (1984); Good et al., in L. H. Lee (ed.) Fundamentals of Adhesion, Plenum, N.Y., chapter 4 (1989)) (FIG. 1). In practice, some proteins do bind more efficiently to the treated hydrophilic polystyrene than to the untreated material. Covalent binding to polystyrene, especially microtiter wells, has however proven to be difficult, so passive adsorption remains the most commonly used method of binding macromolecules to such wells. The term xe2x80x9cpolystyrenexe2x80x9d may also be used to describe styrene-containing copolymers such as: styrene/divinyl benzene, styrene/butadiene, styrene/vinyl benzyl chloride and others.
While polystyrene is an organic hydrophobic substrate, glass provides an inorganic hydrophilic surface. The most common glass format in immunoassays is the microscope slide. Laboratory-grade glasses are predominantly composed of SiO2, but they also may contain B2O3, Al2O3 as well as other oxides (FIG. 2). Interfaces involving such materials have thus become a dynamic area of chemistry in which surfaces have been modified in order to generate desired heterogeneous environments or to incorporate the bulk properties of different phases into a uniform composite structure. Our purpose here then is to use organosilanes for tailoring surfaces with chemically reactive groups mercapto (SH) and/or epoxy.
While numerous methods for the attachment of oligonucleotides and proteins on surfaces have been described, the methods are both expensive and time consuming. The reported covalent attachments of pre-made oligonucleotides onto modified glass surfaces have been always using modified oligonucleotides in order to increase reactivity and selectivity of oligonucleotides towards surfaces. Typical modifications involved the introduction of amino groups, or thio groups into 3xe2x80x2- and/or 5xe2x80x2-oligonucleotides. For example, Stimpson et al. (P.N.A.S. 92:6379-6383 (1995)) reported covalent attachment of 3xe2x80x2-amino oligonucleotides onto epoxy silanized surfaces with acid catalysis but with only {fraction (1/10)} the density achieved in this invention. Beattie et al. (Clin. Chem. 41:700-706 (1995)) reported attachment of 3xe2x80x2- and/or 5xe2x80x2-amino oligonucleotides onto epoxy silanized surfaces under elevated temperature. Lamture et al. (Nucleic Acids Res. 22:2121-2125 (1994)) reported the methods for attaching 3xe2x80x2-amino-oligonucleotides onto epoxy silanized slides under 0.1 M KOH. Hetero bifunctional cross-linkages have been used to couple the 3xe2x80x2 or 5xe2x80x2-thio-modified oligonucleotides or amino-modified onto amino-propyl silanized surfaces as reported by Chrisey et al. (Nucleic Acids Res. 24: 303103039 (1996)) and Guo et al. (Nucleic Acids Res. 22: 4556-5465 (1994)). All of these reported methods however, require modified oligonucleotides.
The present invention describes a novel method for immobilizing nucleic acid molecules to a solid-phase by means of a covalent ether or thioether linkage. This simple, two-step method has the specificity and efficiency needed to prepare DNA arrays.
The present invention provides an improved immobilization method that permits the rapid, and inexpensive immobilization of nucleic acid molecules to a solid phase. The invention allows immobilization of oligonucleotides by incubation with a silane-containing or silane-treated solid phase. The immobilized molecules can be used for nucleic acid hybridization assays, sequencing hybridization assays, genetic analyses, combinatorial analyses involving nucleic acids or proteins, and other screening applications such as protein-DNA binding assays.
In detail, the invention provides a method for immobilizing a nucleic acid molecule to a solid phase, the method comprising the steps of:
(A) coating said solid phase with a silane and allowing said silane-coated solid phase to cure; and
(B) coupling unmodified nucleic acid molecules having either a terminal 3xe2x80x2 OH or a terminal 5xe2x80x2 OH to said silane-coated solid phase.
The invention particularly concerns the embodiments of the above method wherein, in step A, the silane is selected from the group consisting of 3-mercapto-propyl-trimethoxysilane and 3-glycidoxy propyl-trimethoxysilane.
The invention further pertains to oligonucleotides coated surfaces useful in genetic analysis and other screening applications such as protein-DNA binding assays.
The invention particularly concerns oligonucleotide arrays comprising epoxy- or mercapto-silane coated surfaces and unmodified oligonucleotides covalently attached to the epoxy or mercapto-silane, such arrays being useful for nucleic acid hybridization assays, sequencing hybridization assays, genetic analyses, combinatorial analyses involving nucleic acids or proteins, and other screening applications such as protein-DNA binding assays. The features of the coated surface enable standardized patterning of unique oligonucleotides onto silane surface coatings.
The invention further pertains to simultaneous patterning of multiple DNA probes in a high density and in a variety of array formats.